Method of Preparing a Heat-Treated Product

ABSTRACT

The formation of acrylamide during heat treatment in the production of a food product is reduced by treating the raw material with an enzyme before the heat treatment. The enzyme is capable of reacting on asparagine or glutamine (optionally substituted) as a substrate or is a laccase or a peroxidase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/352,511 filed on Jan. 18, 2012 (pending, now allowed), which is adivisional of U.S. application Ser. No. 11/952,640 filed on Dec. 7,2007, now U.S. Pat. No. 8,124,396, which is a continuation of U.S.application Ser. No. 10/530,931 filed on Jun. 14, 2005, now U.S. Pat.No. 7,396,670, which is a 35 U.S.C. 371 national application ofPCT/DK2003/000684 filed on Oct. 10, 2003, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2002 01547filed on Oct. 11, 2002 and U.S. provisional application No. 60/421,897filed on Oct. 29, 2002, the contents of which are fully incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of preparing a heat-treatedproduct with a low water content from raw material comprisingcarbohydrate, protein and water. It also relates to an asparaginase foruse in the method.

BACKGROUND OF THE INVENTION

Tabeke et al. (J. Agric. Food Chem., 2002, 50: 4998-5006) reported thatacrylamide is formed during heating of starch-rich foods to hightemperatures. The acrylamide formation has been ascribed to the Maillardreaction (Mottram et al. and Stadtler et al., 2002, Nature 419:448-449).

WO 00/56762 discloses expressed sequence tags (EST) from A. oryzae. Kimet al., 1988, Asparaginase II of Saccharomyces cerevisiae.Characterization of the ASP3 gene. J. Biol. Chem. 263:11948, disclosesthe peptide sequence of an extra-cellular asparaginase.

SUMMARY OF THE INVENTION

According to the invention, the formation of acrylamide during heattreatment of raw material comprising carbohydrate, protein and water isreduced by treating the raw material with an enzyme before the heattreatment. Accordingly, the invention provides a method of preparing aheat-treated product, comprising the sequential steps of:

a) providing a raw material which comprises carbohydrate, protein andwater

b) treating the raw material with an enzyme, and

c) heat treating to reach a final water content below 35% by weight.

The enzyme is capable of reacting on asparagine or glutamine (optionallysubstituted) as a substrate or is a laccase or a peroxidase.

The invention also provides an asparaginase for use in the process and apolynucleotide encoding the asparaginase.

DETAILED DESCRIPTION OF THE INVENTION Raw Material and Enzyme TreatmentThe raw material comprises carbohydrate, protein and water, typically inamounts of 10-90% or 20-50% carbohydrate of the total weight. Thecarbohydrate may consist mainly of starch, and it may include reducingsugars such as glucose, e.g., added as glucose syrup, honey or drydextrose. The protein may include free amino acids such as asparagineand glutamine (optionally substituted).

The raw material may include tubers, potatoes, grains, oats, barley,corn (maize), wheat, nuts, fruits, dried fruit, bananas, sesame, ryeand/or rice.

The raw material may be in the form of a dough comprising finely dividedingredients (e.g., flour) with water. The enzyme treatment may be doneby mixing (kneading) the enzyme into the dough and optionally holding tolet the enzyme act. The enzyme may be added in the form of an aqueoussolution, a powder, a granulate or agglomerated powder. The dough may beformed into desired shapes, e.g., by sheeting, cutting and/or extrusion.

The raw material may also be in the form of intact vegetable pieces,e.g., slices or other pieces of potato, fruit or bananas, whole nuts,whole grains etc. The enzyme treatment may comprise immersing thevegetable pieces in an aqueous enzyme solution and optionally applyingvacuum infusion. The intact pieces may optionally be blanched byimmersion in hot water, e.g., at 70-100° C., either before or after theenzyme treatment.

The raw material may be grain intended for malting, e.g., malting barleyor wheat. The enzyme treatment of the grain may be done before, duringor after the malting (germination).

The raw material before heat treatment typically has a water content of10-90% by weight and is typically weakly acidic, e.g., having a pH of5-7.

Heat Treatment

The process of the invention involves a heat treatment at hightemperature to reach a final water content (moisture content) in theproduct below 35% by weight, typically 1-20%, 1-10% or 2-5%. During theheat treatment, the temperature at the surface of the product may reach110-220° C., e.g., 110-170° C. or 120-160° C.

The heat treatment may involve, frying, particularly deep frying in tri-and/or di-glycerides (animal or vegetable oil or fat, optionallyhydrogenated), e.g., at temperatures of 150-180° C. The heat treatmentmay also involve baking in hot air, e.g., at 160-310° C. or 200-250° C.for 2-10 minutes, or hot-plate heating. Further, the heat treatment mayinvolve kilning of green malt.

Heat-Treated Product

The process of the invention may be used to produce a heat-treatedproduct with low water content from raw material containing carbohydrateand protein, typically starchy food products fried or baked at hightemperatures. The heat-treated product may be consumed directly as anedible product or may be used as an ingredient for further processing toprepare an edible or potable product.

Examples of products to be consumed directly are potato products, potatochips (crisps), French fries, hash browns, roast potatoes, breakfastcereals, crisp bread, muesli, biscuits, crackers, snack products,tortilla chips, roasted nuts, rice crackers (Japanese “senbei”), wafers,waffles, hot cakes, and pancakes.

Malt (e.g., caramelized malt or so-called chocolate malt) is generallyfurther processed by mashing and brewing to make beer.

Enzyme Capable of Reacting with Asparagine or Glutamine (OptionallySubstituted) as a Substrate

The enzyme may be capable of reacting with asparagine or glutamine whichis optionally glycosylated or substituted with a peptide at thealpha-amino and/or the carboxyl position. The enzyme may be anasparaginase, a glutaminase, an L-amino acid oxidase, aglycosylasparaginase, a glycoamidase or a peptidoglutaminase.

The glutaminase (EC 3.5.1.2) may be derived from Escherichia coli. TheL-amino acid oxidase (EC 1.4.3.2) capable of reacting with asparagine orglutamine (optionally glycosylated) as a substrate may be derived fromTrichoderma harzianum (WO 94/25574). The glycosylasparaginase (EC3.5.1.26, aspartylglucosaminidase,N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase) may be derivedfrom Flavobacterium meningosepticum. The glycoamidase (peptideN-glycosidase, EC 3.5.1.52) may be derived from Flavobacteriummeningosepticum. The peptidoglutaminase may be peptidoglutaminase I orII (EC 3.5.1.43, EC 3.5.1.44).

The enzyme is used in an amount which is effective to reduce the amountof acrylamide in the final product. The amount may be in the range0.1-100 mg enzyme protein per kg dry matter, particularly 1-10 mg/kg.Asparaginase may be added in an amount of 10-100 units per kg dry matterwhere one unit will liberate 1 micromole of ammonia from L-asparagineper min at pH 8.6 at 37° C.

Asparaginase

The asparaginase (EC 3.5.1.1) may be derived from Saccharomycescerevisiae, Candia utilis, Escherichia coli, Aspergillus oryzae,Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, orPenicillium citrinum. It may have the amino acid sequence shown in SEQID NO: 2 (optionally truncated to residues 27-378, 30-378, 75-378 or80-378), 4, 6, 8, 10, 12 or 13 or a sequence which is at least 90%(particularly at least 95%) identical to one of these. It may beproduced by use of the genetic information in SEQ ID NO: 1, 3, 5, 7, 9or 11, e.g., as described in an example.

Whitehead Institute, MIT Center for Genome Research, Fungal GenomeInitiative has published A. nidulans release 1 and F. graminearumrelease 1 on the Internet atgenome.wi.mit.edu/ftp/distribution/annotation/ under the AspergillusSequencing Project and the Fusarium graminearum Sequencing Project.Preliminary sequence data for Aspergillus fumigatus was published on TheInstitute for Genomic Research website atgenome.wi.mit.edu/ftp/distribution/an notation/.

The inventors inserted the gene encoding the asparaginase from A. oryzaeinto E. coli and deposited the clone under the terms of the BudapestTreaty with the DSMZ—Deutsche Sammlung von Microorganismen andZellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. The depositnumber was DSM 15960, deposited on 6 Oct. 2003.

Alignment and Identity

The enzyme and the nucleotide sequence of the invention may havehomologies to the disclosed sequences of at least 90% or at least 95%,e.g., at least 98%.

For purposes of the present invention, alignments of sequences andcalculation of identity scores were done using a Needleman-Wunschalignment (i.e., global alignment), useful for both protein and DNAalignments. The default scoring matrices BLOSUM50 and the identitymatrix are used for protein and DNA alignments respectively. The penaltyfor the first residue in a gap is −12 for proteins and −16 for DNA,while the penalty for additional residues in a gap is −2 for proteinsand −4 for DNA. Alignment is from the FASTA package version v20u6(Pearson and Lipman, 1988, “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and Pearson, 1990, “Rapid and SensitiveSequence Comparison with FASTP and FASTA”, Methods in Enzymology183:63-98).

Laccase or Peroxidase

The laccase (EC 1.10.3.2) may be of plant or microbial origin, e.g.,from bacteria or fungi (including filamentous fungi and yeasts).Examples include laccase from Aspergillus, Neurospora, e.g., N. crassa,Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes,e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani,Coprinus, e.g., C. cinereus, C. comatus, C. friesii, and C. plicatilis,Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus,Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S.thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita, orCoriolus, e.g., C. hirsutus.

The peroxidase (EC 1.11.1.7) may be from plants (e.g., horseradish orsoybean peroxidase) or microorganisms such as fungi or bacteria, e.g.,Coprinus, in particular Coprinus cinereus f. microsporus (IFO 8371), orCoprinus macrorhizus, Pseudomonas, e.g., P. fluorescens (NRRL B-11),Streptoverticillium, e.g., S. verticillium ssp. verticillium (IFO13864), Streptomyces, e.g., S. thermoviolaceus (CBS 278.66),Streptomyces, e.g., S. viridosporus (ATCC 39115), S. badius (ATCC39117), S. phaeochromogenes (NRRL B-3559), Pseudomonas, e.g., P.pyrrocinia (ATCC 15958), Fusarium, e.g., F. oxysporum (DSM 2672) andBacillus, e.g., B. stearothermophilus (ATCC 12978).

Oxidoreductase Capable of Reacting with a Reducing Sugar as a Substrate

The method of the invention may comprise treating the raw material withan oxidoreductase capable of reacting with a reducing sugar as asubstrate. The oxidoreductase may be an oxidase or dehydrogenase capableof reacting with a reducing sugar as a substrate such as glucose andmaltose.

The oxidase may be a glucose oxidase, a pyranose oxidase, a hexoseoxidase, a galactose oxidase (EC 1.1.3.9) or a carbohydrate oxidasewhich has a higher activity on maltose than on glucose. The glucoseoxidase (EC 1.1.3.4) may be derived from Aspergillus niger, e.g., havingthe amino acid sequence described in U.S. Pat. No. 5,094,951. The hexoseoxidase (EC 1.1.3.5) may be derived from algal species such asIridophycus flaccidum, Chondrus crispus and Euthora cristata. Thepyranose oxidase may be derived from Basidiomycete fungi, Peniophoragigantean, Aphyllophorales, Phanerochaete chrysosporium, Polyporuspinsitus, Bierkandera adusta or Phlebiopsis gigantean. The carbohydrateoxidase which has a higher activity on maltose than on glucose may bederived from Microdochium or Acremonium, e.g., from M. nivale (U.S. Pat.No. 6,165,761), A. strictum, A. fusidioides or A. potronii.

The dehydrogenase may be glucose dehydrogenase (EC 1.1.1.47, EC1.1.99.10), galactose dehydrogenase (EC 1.1.1.48), D-aldohexosedehydrogenase (EC 1.1.1.118, EC 1.1.1.119), cellobiose dehydrogenase (EC1.1.5.1, e.g., from Humicola insolens), fructose dehydrogenase (EC1.1.99.11, EC 1.1.1.124, EC 1.1.99.11), aldehyde dehydrogenase (EC1.2.1.3, EC 1.2.1.4, EC 1.2.1.5). Another example is glucose-fructoseoxidoreductase (EC 1.1.99.28).

The oxidoreductase is used in an amount which is effective to reduce theamount of acrylamide in the final product. For glucose oxidase, theamount may be in the range 50-20,000 (e.g., 100-10,000 or 1,000-5,000)GODU/kg dry matter in the raw material. One GODU is the amount of enzymewhich forms 1 μmol of hydrogen peroxide per minute at 30° C., pH 5.6(acetate buffer) with glucose 16.2 g/l (90 mM) as substrate using 20min. incubation time. For other enzymes, the dosage may be foundsimilarly by analyzing with the appropriate substrate.

EXAMPLES Media DAP2C-1

11 g MgSO₄.7H₂O

1 g KH₂PO₄

2 g Citric acid, monohydrate

30 g maltodextrin

6 g K₃PO₄.3H₂O

0.5 g yeast extract

0.5 ml trace metals solution

1 ml Pluronic PE 6100 (BASF, Ludwigshafen, Germany)

Components are blended in one liter distilled water and portioned out toflasks, adding 250 mg CaCO3 to each 150 ml portion.

The medium is sterilized in an autoclave. After cooling the following isadded to 1 liter of medium:

23 ml 50% w/v (NH₄)₂HPO₄, filter sterilized

33 ml 20% lactic acid, filter sterilized

Trace Metals Solution

6.8 g ZnCl₂

2.5 g CuSO₄.5H₂O

0.24 g NiCl₂.6H₂O

13.9 g FeSO₄.7H₂O

8.45 g MnSO₄.H₂O

3 g Citric acid, monohydrate

Components are blended in one liter distilled water.

Asparaginase Activity Assay Stock Solutions

50 mM Tris buffer, pH 8.6

189 mM L-Asparagine solution

1.5 M Trichloroacetic Acid (TCA)

Nessler's reagent, Aldrich Stock No. 34, 514-8 (Sigma-Aldrich, St.Louis, Mo. USA)

Asparaginase, Sigma Stock No. A4887 (Sigma-Aldrich, St. Louis, Mo. USA)

Assay Enzyme Reaction:

500 micro-l buffer

100 micro-l L-asparagine solution

350 micro-l water

are mixed and equilibrated to 37° C.

100 micro-l of enzyme solution is added and the reactions are incubatedat 37° C. for 30 minutes.

The reactions are stopped by placing on ice and adding 50 micro-l of 1.5M TCA.

The samples are mixed and centrifuged for 2 minutes at 20,000 g

Measurement of Free Ammonium:

50 micro-l of the enzyme reaction is mixed with 100 micro-l of water and50 micro-l of Nessler's reagent. The reaction is mixed and absorbance at436 nm is measured after 1 minute.

Standard:

The asparaginase stock (Sigma A4887) is diluted 0.2, 0.5, 1, 1.5, 2, and2.5 U/ml.

Example 1 Expression of an Asparaginase from Aspergillus oryzae inAspergillus oryzae

Libraries of cDNA of mRNA from Aspergillus oryzae were generated,sequenced and stored in a computer database as described in WO 00/56762.

The peptide sequence of asparaginase II from Saccharomyces cerevisiae(Kim et al., 1988, Asparaginase II of Saccharomyces cerevisiae.Characterization of the ASP3 gene. J. Biol. Chem. 263:11948), wascompared to translations of the Aspergillus oryzae partial cDNAsequences using the TFASTXY program, version 3.2t07 (Pearson et al.,1997, Genomics 46:24-36). One translated A. oryzae sequence wasidentified as having 52% identity to yeast asparaginase II through a 165amino acid overlap. The complete sequence of the cDNA insert of thecorresponding clone (deposited as DSM 15960) was determined and ispresented as SEQ ID NO: 1, and the peptide translated from thissequence, AoASP, is presented as SEQ ID NO: 2. This sequence was used todesign primers for PCR amplification of the AoASP encoding-gene from DSM15960, with appropriate restriction sites added to the primer ends tofacilitate sub-cloning of the PCR product (primers AoASP7 and AoASP8,SEQ ID NOS: 14 and 15). PCR amplification was performed using ExtensorHi-Fidelity PCR Master Mix (ABgene, Surrey, U.K.) following themanufacturer's instructions and using an annealing temperature of 55° C.for the first 5 cycles and 65° C. for an additional 30 cycles and anextension time of 1.5 minutes.

The PCR fragment was restricted with BamHI and HindIII and cloned intothe Aspergillus expression vector pMStr57 using standard techniques. Theexpression vector pMStr57 contains the same elements as pCaHj483 (WO98/00529), with minor modifications made to the Aspergillus NA2 promoteras described for the vector pMT2188 in WO 01/12794, and has sequencesfor selection and propogation in E. coli, and selection and expressionin Aspergillus. Specifically, selection in Aspergillus is facilitated bythe amdS gene of Aspergillus nidulans, which allows the use of acetamideas a sole nitrogen source. Expression in Aspergillus is mediated by amodified neutral amylase II (NA2) promoter from Aspergillus niger whichis fused to the 5′ leader sequence of the triose phosphate isomerase(tpi) encoding-gene from Aspergillus nidulans, and the terminator fromthe amyloglucosidase-encoding gene from Aspergillus niger. Theasparaginase-encoding gene of the resulting Aspergillus expressionconstruct, pMStr90, was sequenced and the sequence agreed completelywith that determined previously for the insert of DSM 15960.

The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformed withpMStr90 using standard techniques (Christensen et al., 1988,Biotechnology 6, 1419-1422). Transformants were cultured in DAP2C-1medium shaken at 200 RPM at 30° C. and expression of AoASP was monitoredby SDS-PAGE and by measuring enzyme activity.

Example 2 Purification of Asparaginase

Culture broth from the preceding example was centrifuged (20000×g, 20min) and the supernatants were carefully decanted from the precipitates.The combined supernatants were filtered through a Seitz EKS plate inorder to remove the rest of the Aspergillus host cells. The EKS filtratewas transferred to 10 mM Tris/HCl, pH 8 on a G25 sephadex column andapplied to a Q sepharose HP column equilibrated in the same buffer.After washing the Q sepharose HP column extensively with theequilibration buffer, the asparaginase was eluted with a linear NaClgradient (0→0.5 M) in the same buffer. Fractions from the column wereanalyzed for asparaginase activity (using the pH 6.0 Universal buffer)and fractions with activity were pooled. Ammonium sulfate was added tothe pool to 2.0 M final concentration and the pool was applied to aPhenyl Toyopearl S column equilibrated in 20 mM succinic acid, 2.0 M(NH₄)₂SO₄, pH 6.0. After washing the Phenyl column extensively with theequilibration buffer, the enzyme was eluted with a linear (NH₄)₂SO₄gradient (2.0→0 M) in the same buffer. Fractions from the column wereagain analyzed for asparaginase activity and active fractions werefurther analyzed by SDS-PAGE. Fractions, which were judged only tocontain the asparaginase, were pooled as the purified preparation andwere used for further characterization. The purified asparaginase washeterogeneously glycosylated judged from the coomassie stained SDS-PAGEgel and in addition N-terminal sequencing of the preparation revealedthat the preparation contained different asparaginase forms, as fourdifferent N-termini were found starting at amino acids A₂₇, S₃₀, G₇₅ andA₈₀ respectively of SEQ ID NO: 2. However, the N-terminal sequencingalso indicated that the purified preparation was relatively pure as noother N-terminal sequences were found by the analysis.

Example 3 Properties of Asparaginase

The purified asparaginase from the preceding example was used forcharacterization.

Asparaginase Assay

A coupled enzyme assay was used. Asparaginase was incubated withasparagine and the liberated ammonia was determined with an Ammonia kitfrom Boehringer Mannheim (cat. no. 1 112 732) based on glutamatedehydrogenase and NADH oxidation to NAD⁺ (can be measured as a decreasein A₃₇₅). Hence the decrease in absorbance at 375 nm was taken as ameasure of asparaginase activity.

Asparagine 10 mg/ml L-asparagine (Sigma A-7094) was dissolved substrate:in Universal buffers and pH was adjusted to the indicated pH-values withHCl or NaOH. Temperature: controlled Universal 100 mM succinic acid, 100mM HEPES, 100 mM buffers: CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl,0.01% Triton X-100 adjusted to pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,8.0, 9.0, 10.0, 11.0 and 12.0 with HCl or NaOH. Stop reagent: 500 mM TCA(Trichloroacetic acid). Assay buffer: 1.0 M KH₂PO₄/NaOH, pH 7.5. Ammonia1 NADH tablet + 1.0 ml Bottle 1 (contain 2-oxoglutarate reagent A:(second substrate) and buffer) + 2.0 ml Assay buffer. Ammonia 40 micro-IBottle 3 (contain glutamate dehydrogenase) + reagent B: 1460 micro-IAssay buffer.

450 micro-l asparagine substrate was placed on ice in an Eppendorf tube.50 micro-l asparaginase sample (diluted in 0.01% Triton X-100) wasadded. The assay was initiated by transferring the Eppendorf tube to anEppendorf thermomixer, which was set to the assay temperature. The tubewas incubated for 15 minutes on the Eppendorf thermomixer at its highestshaking rate (1400 rpm). The incubation was stopped by transferring thetube back to the ice bath and adding 500 micro-l Stop reagent. The tubewas vortexed and centrifuged shortly in an icecold centrifuge toprecipitate the proteins in the tube. The amount of ammonia liberated bythe enzyme was measured by the following procedure: 20 micro-lsupernatant was transferred to a microtiter plate, 200 micro-l Ammoniareagent A was added and A₃₇₅ was read (A₃₇₅(initial)). Then 50 micro-lAmmonia reagent B was added and after 10 minutes at room temperature theplate was read again (A₃₇₅(final)). A₃₇₅(initial)−A₃₇₅(final) was ameasure of asparaginase activity. A buffer blind was included in theassay (instead of enzyme) and the decrease in A₃₇₅ in the buffer blindwas subtracted from the enzyme samples.

pH-Activity, pH-Stability, and Temperature-Activity of Asparaginase

The above asparaginase assay was used for obtaining the pH-activityprofile, the pH-stability profile as well as the temperature-activityprofile at pH 7.0. For the pH-stability profile the asparaginase wasdiluted 7× in the Universal buffers and incubated for 2 hours at 37° C.After incubation the asparaginase samples were transferred to neutralpH, before assay for residual activity, by dilution in the pH 7Universal buffer.

The results for the: pH-activity profile at 37° C. were as follows,relative to the residual activity at after 2 hours at pH 7.0 and 5° C.:

pH Asparaginase 2 0.00 3 0.01 4 0.10 5 0.53 6 0.95 7 1.00 8 0.66 9 0.2210 0.08 11 0.00

The results for the pH-stability profile (residual activity after 2hours at 37° C.) were as follows:

pH Asparaginase 2.0 0.00 3.0 0.00 4.0 1.06 5.0 1.08 6.0 1.09 7.0 1.098.0 0.92 9.0 0.00 10.0 0.00 11.0 0.00 12.0 0.00 1.00

The results for the temperature activity profile (at pH 7.0) were asfollows:

Temp (° C.) Asparaginase 15 0.24 25 0.39 37 0.60 50 0.81 60 1.00 70 0.18

Other Characteristics

The relative molecular weight as determined by SDS-PAGE was seen as abroad band (a smear) at M_(r)=40-65 kDa.

N-terminal sequencing showed four different terminals, corresponding toresidues 27-37, 30-40, 75-85 and 80-91 of SEQ ID NO: 2, respectively.

Example 3 Cloning of Asparaginase from Penicillium Citrinum

Penicillium citrinum was grown in MEX-1 medium (Medium B in WO 98/38288)in flasks shaken at 150 RPM at 26° C. for 3 and 4 days. Mycelium washarvested, a cDNA library constructed, and cDNAs encoding secretedpeptides were selected and sequenced by the methods described in WO03/044049. Comparison to known sequences by methods described in WO03/044049 indicated that the Penicillium sequence ZY132299 encoded anasparaginase. The complete sequence of the corresponding cDNA wasdetermined and is presented as SEQ ID NO: 11, and the peptide translatedfrom this sequence is presented as SEQ ID NO: 12.

Example 4 Effect of Asparaginase on Acrylamide Content in Potato Chips

Asparaginase from A. oryzae having the amino acid sequence shown in SEQID NO: 2 was prepared and purified as in Examples 1-2 and added atvarious dosages to potato chips made from 40 g of water, 52.2 g ofdehydrated potato flakes, 5.8 g of potato starch and 2 g of salt.

The flour and dry ingredients were mixed for 30 sec. The salt and enzymewere dissolved in the water, and the solution was adjusted to 30° C. Thesolution was added to the flour. The dough was further mixed for 15 min.The mixed dough was placed in a closed plastic bag and allowed to restfor 15 min at room temperature.

The dough was then initially compressed for 60 sec in a dough press.

The dough was sheeted and folded in a noodle roller machine until anapprox. 5-10 mm dough is obtained. The dough was then rolled around arolling pin and allowed to rest for 30 min in a plastic bag at roomtemperature. The dough was sheeted further to a final sheet thickness ofapprox 1.2 mm.

The sheet was cut into squares of approx 3×5 cm.

The sheets were placed in a frying basket, placed in an oil bath andfried for 45 sec at 180° C. The noodle basket was held at a 45° angleuntil the oil stopped dripping. The products were removed from thebasket and left to cool on dry absorbent paper.

The potato chips were homogenized and analyzed for acrylamide. Theresults were as follows:

Asparaginase dosage Acrylamide U/kg potato dry matter Micro-g per kg 05,200 100 4,600 500 3,100 1000 1,200 2000 150

The results demonstrate that the asparaginase treatment is effective toreduce the acrylamide content in potato chips, that the acrylamidereduction is clearly dosage dependent, and that the acrylamide contentcan be reduced to a very low level.

Example 5 Effect of Various Enzymes on Acrylamide Content in PotatoChips

Potato chips were made as follows with addition of enzyme systems whichare capable of reacting on asparagine, as indicated below.

Recipe:

Tap water 40 g Potato flakes dehydrated 52.2 g Potato starch 5.8 g Salt2 g

Dough Procedure:

The potato flakes and potato starch are mixed for 30 sec in a mixer atspeed 5. Salt and enzyme are dissolved in the water. The solution isadjusted to 30° C.+/−1° C. Stop mixer, add all of the salt/enzymesolution to flour. The dough is further mixed for 15 min.

Place mixed dough in plastic bag, close bag and allow the dough to restfor 15 min at room temperature.

The dough is then initially compressed for 60 sec in a dough press.

The dough is sheeted and folded in a noodle roller machine until anapprox. 5-10 mm dough is obtained. The dough is then rolled around arolling pin and the dough is allowed to rest for 30 min in a plastic bagat room temperature. The dough is sheeted further to a final sheetthickness of approx 1.2 mm.

Cut the sheet into squares of approx 3×5 cm.

Sheets are placed in a frying basket, placed in the oil bath and friedfor 60 sec at 180° C. Hold the noodle basket at a 45° angle and let theproduct drain until oil stops dripping. Remove the products from thebasket and leave them to cool on dry absorbent paper.

The results from the acrylamide analysis were as follows:

Enzyme dosage per kg Acrylamide Enzyme of potato dry matter Micro-g perkg None (control) 0 4,100 Asparaginase from 1000 U/kg 150 ErwiniaChrysanthemi A-2925 Glutaminase 50 mg enzyme 1,800 (product of Daiwa)protein/kg Amino acid oxidase from 50 mg enzyme 1,300 Trichodermaharzianum protein/kg described in WO 9425574. Laccase from 5000LAMU/kg + 2,000 Myceliophthora thermophila + 75 mg enzyme peroxidasefrom Coprinus protein/kg

The results demonstrate that all the tested enzyme systems are effectivein reducing the acrylamide content of potato chips.

1-11. (canceled)
 12. A method of preparing a heat-treated product,comprising the sequential steps of: a) providing a raw material whichcomprises carbohydrate, protein and water, b) treating the raw materialwith an asparaginase, and c) heat treating to reach a final watercontent below 35% by weight, wherein the heat treatment involves fryingat temperatures of 150-180° C., baking in hot air at 160-310° C.,hot-plate heating and/or kilning of green malt.
 13. The method of claim12, which further comprises treating the raw material with anoxidoreductase capable of reacting with a reducing sugar as a substrate.14. The method of claim 13, wherein the oxidoreductase capable ofreacting with a reducing sugar as a substrate is a glucose oxidase; apyranose oxidase; a hexose oxidase; a galactose oxidase; or acarbohydrate oxidase which has a higher activity on maltose than onglucose.
 15. The method of claim 12, wherein the raw material is in theform of a dough and the enzyme treatment comprises mixing the enzymeinto the dough.
 16. The method of claim 12, wherein the raw materialcomprises intact vegetable pieces and the enzyme treatment comprisesimmersing the vegetable pieces in an aqueous solution of the enzyme. 17.The method of claim 12, wherein the raw material comprises a potatoproduct.
 18. The method of claim 12, wherein the asparaginase has anamino acid sequence which is at least 90% identical to SEQ ID NO: 2(optionally truncated to residues 27-378, 30-378, 75-378 or 80-378), 4,6, 8, 10, 12 or
 13. 19. The method of claim 12, wherein the heat-treatedproduct is selected among a potato product, potato chips, potato crisps,French fries, hash browns, roast potatoes, breakfast cereals, crispbread, muesli, biscuits, crackers, snack products, tortilla chips,roasted nuts, rice crackers, Japanese “senbei”, wafers, waffles, hotcakes, and pancakes.
 20. The method of claim 12, wherein theheat-treated product is a potato product.
 21. The method of claim 12,wherein the heat-treated product is French fries.